Antibody-Mediated Rejection in Kidney Transplantation

FDA Public Workshop: Antibody-mediated Rejection in Kidney Transplantation

The Choice of Induction / Maintenance Immunosuppression and their Impact on Preexisting and De Novo Antibodies

Millie Samaniego, MD, FACP, FASN, FAST Alan B. Leichtman Collegiate Chair of Transplant Nephrology Professor of Medicine University of Michigan Disclosures

• I have nothing to disclose in relation to this presentation: • Following drugs used “off label” will be discussed: • Thymoglobulin • Campath • IVIg • Belatacept • Rituximab • Eculizumab • C1q Inhibitors TABLE 1

The Treatment of Acute Antibody-Mediated Rejection in Kidney Transplant Recipients-A Systematic Review. Roberts, Darren; Jiang, Simon; Chadban, Steven

Transplantation. 94(8):775-783, October 27, 2012. DOI: 10.1097/TP.0b013e31825d1587

TABLE 1 Therapeutic agents used against DSAs in the treatment of antibody- mediated rejection and the evidence supporting their role

The Treatment of Acute Antibody-Mediated Rejection in Kidney Transplant Recipients-A Systematic Review. Roberts, Darren; Jiang, Simon; Chadban, Steven

Transplantation. 94(8):775-783, October 27, 2012. DOI: 10.1097/TP.0b013e31825d1587

FIGURE 2 . Trends in the use of treatments for AMR over time on the basis of all publications identified in the systematic review. Using a gray scale, black represents the most commonly used (in the case of PP, approximately 700 patients were treated between 2007 and 2011), whereas no color (white) means that it was not used. This includes patients from any observational, treatment, or epidemiology-based study identified in the systematic review. The use of tacrolimus and mycophenolate in patients with AMR is likely to be underrepresented in this figure because in recent years, these treatments are commonly used as baseline immunosuppression for high-risk KTRs, whereas the data included here only included new treatments administered to patients in response to a diagnosis of AMR/vascular rejection.

• Suppression of the T-cell response • Elimination of pre -formed antibody • Inhibition of residual antibody and the complement system cascade • IVIg • Eculizumab • C1q inhibitors • Depletion of antibody producing cells and/or their precursors • Rituximab ABMR Protocols

•Suppression of the T -cell response • Depletional Agents • Polyclonal: rATG • Humanized antibodies: Alemtuzumab • Non-depletional Agents • Belatacept Tra n sp lantati on 9 7 ( 6) March 20 14 Methods

• 114 moderately sensitized deceased donor renal transplant recipients between December 2009 and November 2011 • Positive DSA (MFI max 500-4,000 by SAB Luminex) • Negative flow crossmatch • Induction: ATG or basiliximab based on provider preference • Maintenance: TAC/MPA/Pred • Followed for 36 months • De novo DSA • Defined as the absence of pre-transplant DSA (MFI less than 300) increasing at least three -fold (greater than 900 MFI) post-transplant • Assessed blindly by HLA lab director (TE)

Summary and Conclusion

• Induction with ATG (thymoglobulin) is associated with a reduction in the incidence of dnDSA and ABMR without a significant change in the incidence of CMV infection or BK nephropathy when compared with basiliximab in moderately sensitized deceased donor renal transplant recipients • Randomized clinical trials are required to specifically address the role of induction immunosuppression on dnDSA and ABMR ALEMTUZUMAB (CAMPATH-1H)

• A recombinant DNA- derived humanized monoclonal antibody directed against CD52 • IgG1 kappa with human variable framework and constant regions with murine CDR regions Uses of Alemtuzumab in Kidney Transplantation

• Antibody-Induction Agent: • CNI avoidance • Steroid-free protocols • CNI monotherapy • “Prope” tolerance induction • Induction of HIV patients undergoing KTx • Induction in desensitization protocols • Treatment of Acute Rejection: • Four studies encompassing an N=62 • Two studies used Campath -1H and one Campath-1G • Two published in a peer-reviewed journal De Novo a-HLA Antibodies in Alemtuzumab Induction

• Cai J et al: Transplantation 2004; 78:919 -924 • 42% (n=10) of the patients enrolled in the pilot study of alemtuzumab induction for CNI avoidance have developed Class I and Class II a -HLA antibodies • 60% DSA • 40% non-DSA • At 24 mos of F/U: • Of 10 Pts developing a-HLA antibodies, 4 (40%) have had BPAMR • Of these: • 3 patients had DSA a-HLA antibodies • 1 patient had non-DSA a-HLA antibodies

Alemtuzumab Induction in Renal Transplantation

Michael J. Hanaway, M.D., E. Steve Woodle, M.D., Shamkant Mulgaonkar, M.D., V. Ram Peddi, M.D., Dixon B. Kaufman, M.D., Ph.D., M. Roy First, M.D., Richard Croy, M.A., John Holman, M.D., for the INTAC Study Group

N Engl J Med Volume 364(20):1909-1919 May 19, 2011

J Am Soc Nephrol 28: 2017

Rituximab+IVIg: Final Verdict?

• 2/11-6/12: • Randomized to IVIg+Placebo or IVIg+Ritux • Total enrollment goal = 90 patients • End-points: • Rates of Transplantation • ABMR • Protocol biopsies at 1 year • Scr, DSA monitoring • Patient and Graft survival

Transplantation. 98(3):312-319, August 15, 2014. DOI: 10.1097/TP.0000000000000064 FIGURE 1

Benefits of Rituximab Combined With Intravenous Immunoglobulin for Desensitization in Kidney Transplant Recipients. Vo, Ashley; Choi, Jua ; Cisneros, Kristen; Reinsmoen, Nancy; Haas, Mark; Ge, Shili ; Toyoda, Mieko; Kahwaji, Joseph; Peng, Alice; Villicana, Rafael; Jordan, Stanley

Transplantation. 98(3):312-319, August 15, 2014. DOI: 10.1097/TP.0000000000000064

FIGURE 1 . Study design. Patients were randomized in a 1:1 ratio to receive IVIG+placebo versus IVIG+rituximab. Treatment is as described in the text. The attribution to study groups is shown. Briefly, 15 patients were enrolled. Of those, 87% underwent transplantation. Seven patients were randomized to IVIG+placebo group; six patients were randomized to IVIG+rituximab group. Outcomes are described in the text.

© 2014 by Lippincott Williams & Wilkins. Published by LippincottTransplantation. Williams & Wilkins, Inc. 98(3):312-319, August2 15, 2014. DOI: 10.1097/TP.0000000000000064 Outcomes End-points Blinded study IVIg IVIg+Ritux Enrollment 15 Transplant 11 (73%) 5 6 Rate ABMR 3 3 (43%) 0 Graft loss 2 2 0 Cause of GL ABMR, BKVN 0

Rx of ABMR required PLEX+Ritux+Eculizumab Efficacy similar in enabling transplantation by reduction of DSA DSA monitoring showed rebound at 6 mo in the IVIg placebo group associated with 60% severe AMBR (3/6) IVIg+Ritux patients (50%; 3/6) had no ABMR in protocol Bx

clinical investigation AM Jackson et al.: Rituximab induction and HLA antibody production

transplantation using a desensitization protocol that transplant. In patients transplanted without rituximab, more either did or did not include a single dose of rituximab HLA antibodies were increased at 1 month post transplant, (375 mg/m2) the day before transplantation. Patient demo- 32% (eight of 25) of DSAs and 55% (50 of 91) of non-DSAs. graphics are provided in Table 1 and reflect our practice of The frequency of HLA antibody rebound was significantly using rituximab for patients with a higher risk for AMR.27,28 higher in patients transplanted without rituximab induction The 25 patients who received rituximab induction had for both DSA, P 0.03, and non-DSA, P 0.003. Moreover, broader sensitization (mean calculated panel reactive anti- the magnitude of¼ the antibody increase¼ was also larger in body (CPRA) 80 vs. 60%, P 0.02), a higher incidence of patients transplanted without rituximab induction. The previous transplants¼ (76 vs.¼ 28%, P 0.002), and repeat mean percentage increase at 1 month for all HLA antibodies ¼ HLA mismatches (80 vs. 0%, Po0.0001). However, the two examined was 294 (median 70) among the lower immu- cohorts had similar DSA levels before desensitization and nologic risk patients transplanted¼ without rituximab, com- received a similar number of plasmapheresis treatments pared with 207 (median 10) for those transplanted with (Table 1, P 0.20). rituximab induction (P ¼0.02). HLA antibody¼ monitoring within the first 2 weeks post In both cohorts, HLA¼ antibody rebounds detected at transplant revealed an increase in DSA for 36% (nine of 25) 1–3 months post transplant were transient, with 95 of 105 of rituximab-treated patients and 44% (11 of 25) of non- antibodies (90%) subsiding without any further plasmaphe- treated patients transplanted without rituximab (P 0.77). resis treatments. Of the 10 DSAs detected at 1 month post Elevated DSA was treated with continued plasmapheresis¼ and transplant, seven remained at a low level detectable only by low-dose intravenous immunoglobulin (IVIg); however, all bead immunoassays. The remaining three DSAs were of patients completed desensitization treatments within 2 weeks moderate levels, detected at a level sufficient to yield a weakly of transplant. An extended analysis was performed on 256 positive FCXM. These stronger rebounds occurred in three HLA antibodies (DSA and non-DSA) to examine HLA patients, two of whom received rituximab induction. antibody levels following the cessation of plasmapheresis/ Protocol biopsy surveillance at 1 month identified subclinical IVIg treatments. The percentage change, comparing HLA rejection (histopathology evidence of AMR without graft antibody levels before desensitization (time zero) with four dysfunction) in seven patients with DSA rebound, including time points (1, 3, 6, and 12 months) post transplant, is the three patients with the strongest DSA levels. Two patients plotted in Figure 1. The mean fluorescence intensity (MFI) showed no evidence of rejection and one patient, with for each antibody was normalized to the positive control bead normal allograft function, was not biopsied because of value, to account for inter-run variability, and the percentage anticoagulation therapy. change from time zero was calculated. Among rituximab- No distinction between HLA class I vs. class II specificities AM Jackson et al.: Rituximab induction and HLA antibody production treated patients, 7% (two of 29) of DSAs examined andclinical 33% was observed investigation in antibodies (DSA or non-DSA) that increased (37 of 111) of non-DSAs were increased at 1 month post post transplantation. For rituximab-treated patients, the these cohorts.Jackson The AM number et al of Kidney cell-mediated Int 2014 [E rejections Pub] was a 20 Rituximab treated higher in the rituximab-treated cohort, which may be due Non-rituximab treated to increased allo-sensitization in this cohort or a consequence 15 ofTable deleting 1 | Patient demographics B-regulatory cells.29,30 Our study is the first to determine the impact of rituximab 10 induction on DSA reboundRituximab in N patient 25 cohortsNo rituximab N receiving 25 P-value the same desensitization regimen without additional B-cell–

Percent died ¼ ¼ depleting therapy.14,18,23,25 Gloor et al. reported decreased 5 ± ± butRecipient persistentage (mean, s.d.) DSA in 10 of41 1215 HLA-incompatible48 13 renal 0.08 recipientsMale gender (no. of patients, at 4 %) months post transplant.8 (32%) These recipients7 (28%) were 1.0 0 P =0.62 desensitizedPrevious Txn (no. of patients, using %) a protocol that19 (76%) included plasmapheresis,7 (28%) 0.002 0 1 2 3 4 5 IVIg, and a single dose of rituximab (375 mg/m2) as induction; Years since transplantation Previous Txn X3 5 (20%) 0 0.06 Number at risk however, all recipients also underwent splenectomy Rituximab treated 25 24 24 19 13 11 beforeHLA-A;B;DR;DQ mismatch transplant.(mean) Studies by4.8 Hirai et al., Takagi5.0 et al., 0.61 Non-rituximab treated 25 24 24 23 17 12 andRepeat HLA Loupy mismatch (No. etof patients, al. %) compared post-transplant20 (80%) DSA0 levels in 0.0001 a b 25 HLA-incompatibleCDC CPRA (mean, median) recipients transplanted48, 50 following26, 3 desen- 0.02 Rituximab treated sitization, which included plasmapheresis and rituximab Non-rituximab treated FCXM CPRA (mean, median) 80, 89 60, 60 0.02 20 induction, with retrospective cohorts transplanted across DSA with no desensitization, or with deceased 15 donorCrossmatch strength transplants (no. of patients) using a protocol involving high-dose IVIgCDC alone. 21 1.0 10 FCXMHLAþ antibody rebounds observed9 at 1–3 months11 post 0.77 Percent graft loss transplantationþ (Figure 1) were transient and declined 5 FCXM , DSA 14 13 1.0 withoutÀ þ intervention, suggesting that they were generated by newly formed short-lived plasma cells.31,32 Analysis of P =0.23 0 bothNumber of DSADSAsb (mean, and median) non-DSA levels post2.0, 2.0 transplantation1.7, 1.0 confirms 0.59 0 1 2 3 4 5 that the trauma of surgery and antigenic stimulation of Years since transplantation Donor age (mean, s.d.) 38±12 46±11 0.03 Number at risk the immune system can elicit both antigen-specific and Rituximab treated 25 24 24 18 12 10 nonspecificNo. of pre-transplant plasmapheresis antibody (mean) responses333.7 and that the current2.3 B-cell 0.08 Non-rituximab treated 25 23 23 22 15 10 depletionNo. of post-transplant strategiesplasmapheresis (mean) can reduce or4.1 blunt but not prevent3.9 new 0.81 Figure 4 | Mortality and death-censored graft loss in the HLA antibody production post transplant. Reducing B-cell rituximab-treated versus non-rituximab–treated cohorts. Anti-CD25 induction (no. of patients, %) 10 (40%) 12 (48%) 0.78 numbers in sensitized patients before the transplant likely Kaplan–Meier estimates of (a) the patients’ 1- and 5-year survival were Thymoglobulin induction (no. of patients, %) 15 (60%) 13 (52%) 0.78 96.0% (95% CI: 74.8–99.4%) and 81.7% (95% CI: 51.9–94.0%) in the decreased the number of HLA-specific B cells that could be rituximab-treated group and 96.0% (95% CI: 74.8–99.4%) and 83.7% activatedAbbreviations: CDC, complement-dependent and transformed cytotoxicity; CPRA, calculated into panel reactive antibody-producing antibody; DSA, donor-specific antibodies; FCXM, flow plasma cytometric crossmatch; HLA, (95% CI: 56.3–94.6%) in the non-rituximab-treated group (P 0.6); cells. Therefore, in the rituximab-treated recipients, the new (b) graft 1 and 5-year death-censored survival were 100.0 and¼ 94.7% human leukocyte antigen; Txn, transplantation. (95% CI: 68.0–99.0%) in the rituximab-treated group and 95.8% (95% plasmaaCPRA was determined cells for HLA-specific likely antibodies of arosesufficient strength from to yield a positive memory cytotoxicity (CDC) or FCXM. B cells residing in secondary lymphoid niches and protected from deple- CI: 73.9–99.4%) and 77.5% (95% CI: 49.7–91.1%) in the non-rituximab bNumber of DSAs before desensitization. treated group (P 0.23). CI, confidence interval. 34–38 Examination of the HLA antibody specificities that ¼ tion. increased post transplantation in our rituximab-treated patients showed that 34 of 39 were to immunogenic responses because of higher CPRAs, multiple transplants, and epitopes2 the patient had been previously exposed to inKidney International repeated HLA mismatches; yet, they experienced less HLA previous allografts or pregnancies and support the existence antibody rebound (DSA and non-DSA). This reduction in of these immune memory reservoirs. These findings suggest a HLA humoral responses, however, did not translate into reduced benefit in avoiding repeated HLA antigen mismatches in rates of AMR when compared with patients transplanted patients with previous transplants or HLA antigens shared without rituximab. Rituximab induction did not significantly with spouses when transplanting multiparous women, to affect the persistence of DSA at 1 year post transplantation, guard against reactivation of occult sensitization. In such which was detected in 52% of the patients treated with cases, kidney paired donation may offer a mechanism to rituximab vs. 40% in the non-rituximab-treated group. This increase HLA matching between recipient–donor pairs and incidence of DSA persistence following rituximab induction avoid repeated HLA mismatches. was higher in our study compared with reports by Loupy The increase in some, but not all, HLA antibody specifi- et al. (28.5%)14 or Takagi et al. (14%)25 at 1 year post cities may reflect differences in the amount of B-cell memory transplantation and likely reflects the increased breadth and that exists toward a particular HLA antigen. We have strength of HLA sensitization in our patients. No significant previously shown that the frequency of peripheral blood, difference in renal function was observed at 1 and 2 years HLA-specific B cells, measured using HLA tetramers, or graft survival at 5 years post transplantation between reflects prior sensitization to an HLA antigen and predicts

Kidney International 5

Humoral Rejection Rates

Negative X-match N AMR @ Tx High dose IVIG 5 4/5 (80%)

PP/IVIg/anti-CD20 30 11/30 (37%)

PP/IVIg/monitoring 14 4/14 (29%)

Positive X-match @ High dose IVIg 7/1- (70%) Tx PP/IVIg/anti-CD20 10 5/10 (50% GL) Stegall et al: Am J Transplant 2006 2nd Generation CTLA4-Ig (LEA29Y)

• Fusion-Receptor Protein including the extracellular region of human CTLA4 and Fc domain of IgG1 • Increased binding avidity to CD80 (B7.1) and CD86 (B7.2) • 10-fold increase in “ in vitro ” effectiveness in inhibiting T cell effector function than CTLA4 -Ig Acute Rejection: Incidence and Banff Grade

Belatacept MI Belatacept LI CsA (n = 219) (n = 226) (n = 221) Acute rejection, % 22% 17% 7%

Banff 97 grade, % Mild acute (IA) 3 2 1 Mild acute (IB) 1 4 2 Moderate acute (IIA) 8 7 3 Moderate acute (IIB) 9 4 1 Severe acute (III) 1 <1 0 Anti-Donor Antibodies by Month 12

Belatacept Belatacept MI LI CsA (n = 219) (n = 226) (n = 221) No AR Patients with anti -donor 4 / 164 (2%) 3 / 179 (2%) 13 / 184 (7%) HLA positive, n/N (%)

With AR Patients with anti -donor 2 / 44 (5%) 0 / 37 (0) 1 / 14 (7%) HLA positive, n/N (%)

35 Stegall et al.

To assess the presence and amount of DSA, we primarily used cell-based transplant PE. Patients in the control group who received pretransplant PE T- and B-flow cytometric crossmatch channel shift (2,9). T-cell antihuman also underwent 4–14 posttransplant PE treatments. Three patients in the globulin cytotoxicity assays were also performed. The B flow cytometric eculizumab group received posttransplant PE early in the study, but this crossmatch (BFXM) is used primarily by our group because it estimates to- approach was discontinued as it was deemed unnecessary for the preven- tal DSA against both class I and class II HLA. BFXM data are presented here tion of AMR. All patients received induction with antithymocyte globulin as channel shifts using a 1024 linear scale. The channel shift is the observed (Thymoglobulin R ;1.5mg/kg/d 5–7 doses) and maintenance immuno- ⃝ × number of channels (raw fluorescence) above the negative control. We pre- suppression with tacrolimus, mycophenolate mofetil and prednisone as viously have published the correlation between the channel shift in our FXM previously described (9). assays to an independent standard (molecules of equivalent soluble fluo- rochrome units) (2). A solid-phase assay (LABscreen, One Lambda, Canoga Renal allograft biopsies were obtained percutaneously using ultrasound Park, CA, USA) was also used to verify alloantibody specificities and amount guidance, processed for light microscopy and immunofluorescence for per- (reported as the highest mean fluorescence index (MFI) against donor HLA). itubular capillary staining for C4d. All biopsies were reviewed by a patholo- DSA levels were measured at baseline (prior to any therapy), the day prior gist (LDC) in a blinded fashion. AMR was diagnosed using standard Banff to transplant (after multiple PE in patients undergoing pretransplant PE) and criteria (10) in combination with graft dysfunction (increase in serum cre- posttransplant on days 4, 7, 14, 21 and 28. atinine 0.3 mg/dL over nadir). In the control group, all patients had at ≥ least one protocol biopsy in the first month after transplantation (between Patients and treatment protocols posttransplant day 7 and 14) and most also had a 1-year protocol biopsy. Inclusion criteria primarily were age greater than 18 years and a base- Eculizumab-treated patients underwent biopsies on posttransplant days 7, line BFXM against their living donor with a channel shift <450 and 200. 14, 28, 90 and 365. Biopsy findings were correlated with DSA levels per- ≥ The eculizumab group consisted of consecutive recipients transplanted be- formed on serum collected within 24 h of the biopsy. tween January 6, 2008 and January 8, 2010 and a historical control group American Journal of Transplantation 2011; 11: 2405–2413 C Copyright 2011 The American Society of Transplantation Serum creatinine values were⃝ determined almost daily for the first 28 days consistedWiley of Periodicals consecutive transplants Inc. between January 1, 2005 and January and the American Society of Transplant Surgeons 10, 2007 that met the inclusion criteria. A history of a splenectomy was an in all recipients and at least monthly thereafter. exclusion criterion. In both eras, we excluded patients from these protocols doi: 10.1111/j.1600-6143.2011.03757.x who, in the investigators opinion, had severe cardiovascular or pulmonary Eculizumab dosing disease, or otherwise were too sick to undergo positive crossmatch kidney The eculizumab dosing regimen was modified from that used in the treat- transplantation. Note, in both time periods, patients with a baseline BFXM ment of paroxysmal nocturnal hemoglobinuria and consisted of 1200 mg >450Terminal were considered to have DSAComplement levels too high for transplantation inInhibitionimmediately prior to transplantation, Decreases 600 mg on postoperative day 1, and our program and thus were excluded a priori. 600 mg weekly thereafter for 4 weeks (Figure 1). At week 4, assessment of Antibody-Mediated RejectionDSA levels was performed.in Sensitized Eculizumab was discontinued in patients whose Eculizumab group patients were immunized against meningococcus and DSA had significantly decreased (B flow crossmatch channel shift <200). pneumococcusRenal 1 month Transplant prior to transplantation. Otherwise, Recipients pretransplant In patients with persistently high DSA and thus believed to have contin- management was the same in the two groups. Recipients with a channel ued high risk for AMR, eculizumab treatment continued (1200 mg week shift 300 underwent pretransplant PE to achieve both T- and B-flow cross- 5, and then every 2 weeks). Another DSA assessment was performed at ≥ Stegall MD et al Am J Transplant 2011; 11 match channel shifts <300 on the day of transplant (2). Recipients with a week 9 and eculizumab was discontinued if the B flow crossmatch chan- T- andM. B-flow D. crossmatch Stegalla channel,*,T.Diwan shift <300 ata,S.Raghavaiah baseline received no pre-a, nel shift wasKeywords:<200. In three patientsAlloantibodies, who received PE posttransplant, anti-HLA an antibodies, L. D. Cornellb,J.Burnsa,c,P.G.Deana, antibody-mediated rejection, complement, chronic rejection, kidney transplantation, sensitized recipients F. G . C o s i o d, M. J. Gandhib, W. Kremerse d and J. M. Gloor Abbreviations: AMR, antibody-mediated rejection; BFXM, B-cell flow cytometric crossmatch; DSA, donor- a William J. von Liebig Transplant Center, Division of specific alloantibody; HLA, human leukocyte antigens; Transplantation Surgery, Mayo Clinic, Rochester, MN PE, plasma exchange. bDepartment of Anatomic Pathology, Mayo Clinic, Rochester, MN Received 01 March 2011, revised 31 May 2011 and c Department of Surgery, Division of Transplant Surgery, accepted for publication 01 June 2011 University of Cincinnati School of Medicine, Cincinnati, OH dDivision of Nephrology and Hypertension, Mayo Clinic, Rochester, MN Donor-specific alloantibody (DSA) against class I and class II eDivision of Biomedical Statistics and Informatics, Mayo Clinic, Rochester, MN human leukocyte antigens (HLA) remains a major barrier to successful renal transplantation. Using desensitization pro- ∗Corrosponding author: Mark D. Stegall, [email protected] tocols involving multiple plasma exchange (PE) treatments and/or high dose intravenous immunoglobulin (1–3), hyper- acute rejection generally has been avoided. However, early Sensitized renal transplant recipients with high lev- antibody-mediated rejection (AMR) primarily occurring with Figureels 1: of Eculizumab donor-specific treatment alloantibody protocol. The (DSA) eculizumab commonly dosing regimen de- was modified from that used in the treatment of paroxysmal nocturnalvelop hemoglobinuria antibody-mediated and consisted rejection of 1200 mg (AMR), immediately which prior may to transplantation,the first 600 month mg on after postoperative transplantation day 1 and has then emerged 600 as the mg weeklycause thereafter acute graft for 4 weeks. loss or At shorten week 4, assessment allograft of survival. DSA levels We was performed.next major Eculizumab complication was discontinued with anin patients incidence whose of up to 40% DSAexamined had significantly the decreased efficacy (B of flow terminal crossmatch complement channel shift < inhibi-200). In patientsusing with current persistently protocols high DSA (2,4). and thus AMR believed may to require have aggressive continuedtion with high risk the for humanized AMR, eculizumab anti-C5 treatment antibody, continued eculizumab, (1200 mg week 5,treatment and then every involving 2 weeks). multiple Another PEs DSA and assessment splenectomy to pre- wasin performed the prevention at week 9 and eculizumabAMR in was renal discontinued transplant if the B recipi- flow crossmatchvent channel acute shift graft was loss.<200. Even successfully treated AMR is ents with a positive crossmatch against their living associated with shortened allograft survival (4). 2406donor. The incidence of biopsy-proven AMR in the firstAmerican Journal of Transplantation 2011; 11: 2405–2413 3 months posttransplant in 26 highly sensitized recip- Since the mechanism of AMR appears to involve the acti- ients of living donor renal transplants who received vation of complement in the setting of high levels of DSA eculizumab posttransplant was compared to a histori- (5–7), we hypothesized that blocking terminal complement cal control group of 51 sensitized patients treated with asimilarplasmaexchange(PE)-basedprotocolwithout activation might prevent AMR in highly sensitized renal eculizumab. The incidence of AMR was 7.7% (2/26) in transplant recipients. The current study examined the effi- the eculizumab group compared to 41.2% (21/51) in cacy of terminal complement blockade in the prevention of the control group (p 0.0031). Eculizumab also de- AMR using eculizumab, a humanized IgG2/4k monoclonal creased AMR in patients= who developed high levels antibody with high affinity for C5 (8). of DSA early after transplantation that caused proximal complement activation. With eculizumab, AMR episodes were easily treated with PE reducing the need for splenectomy. On 1-year protocol biopsy, transplant Methods glomerulopathy was found to be present in 6.7% (1/15) eculizumab-treated recipients and in 35.7% (15/42) of control patients (p 0.044). Inhibition of terminal com- Study design plement activation= with eculizumab decreases the in- The study used an open-label design. Patients were enrolled with informed cidence of early AMR in sensitized renal transplant re- consent using protocols approved by the Institutional Review Board of the cipients (ClincalTrials.gov number NCT006707). Mayo Foundation and Clinic (ClincalTrials.gov number, NCT006707). The goal was to examine whether eculizumab could reduce the incidence of AMR in the first 3 months after kidney transplant in sensitized recipients with DSA against their living donor.

2405 Stegall et al.

Ta b l e 2 : Posttransplant outcomes in the eculizumab-treated and control groups Category Eculizumab group (n 26) Control group (n 51) p-Value = = Follow-up 11.8 6.3 48.8 14.1 ± ± (mean months SD, range) (3.0–27.5) (7.8–69.8) ± Graft survival at 1 year (n, %) 16/16 (100%) 49/51 (96%) 1.00 Antibody-mediated rejection 3months (n, %) 2 (7.7%) 21 (41%) 0.0031 ≤ Patients developing high DSA levels 3months1 13 (50%) 22 (43%) 0.63 ≤ High DSA biopsies C4d (n, %) 13 (100%) 20 (91%) 0.52 + High DSA and C4d biopsies showing AMR (n, %) 2 (15%) 20 (100%) <0.0001 + Cellular rejection 3months (n,%) 1(6.2%) 1(2%) 0.42 ≤ Plasma exchange posttransplant Patients receiving PE (n, %) 3 (12%) 39 (76%) <0.0001 Number of PE treatments (mean SD) 0.35 1.1 7.9 7.5 <0.0001 ± ± ± Splenectomy (n, %) 0 (0%) 9 (18%) 0.025 Graft dysfunction in first month (mg/dL) (maximum 0.45 0.37 0.93 1.15 0.05 ± ± serum creatinine – nadir serum creatinine) Histology at 1 year Transplant glomerulopathy incidence (n, %) 1/15 (6.7%) 15/42 (36%) 0.044 Cg score (mean SD) 0.20 0.78 0.74 1.13 0.17 ± ± ± Ci score (mean SD) 1.00 0.76 0.79 0.80 0.31 ± ± ± Ct score (mean SD) 1.13 0.74 0.91 0.80 0.33 ± ± ± Cv score (mean SD) 0.80 0.68 0.59 0.74 0.23 ± ± ± 1B flow crossmatch channel shift >350 at any time point in the first 3 months. solid-phase assay (MFI 7188 3503 in eculizumab vs. staining) was observed in 91% of control and in 100% ± 6473 4946 controls, p 0.51, see Table S1 for detailed of eculizumab-treated patients who developed high levels ± = DSA data). More patients in the eculizumab group had a of DSA early after transplantation. In the control group, previous transplant compared to controls (50% (13/26) vs. all patients with high levels of DSA and C4d staining + 25%, (13/51); p 0.043). No patient was lost to follow-up (20/20) showed AMR while only 15% of patients (2/13) in = or removed from the study for any reason. the eculizumab group with this combination showed AMR (p < 0.001). All cases of AMR in both groups were ac- companied by an increase in serum creatinine from base- Patient and graft survival line of >0.3 mg/dL and thus would be considered clinical All patients had at least 3 months follow-up (eculizumab AMR. One patient in the control group with an increase of group, mean of 11.9 6.1 months, range 3.0–27.5; = ± 0.2 mg/dL showed clear evidence of AMR on biopsy and control group, mean 48.8 14.1 months, range 7.8– = ± thus was designated as AMR. 69.8) (Table 2). In the historical control group, one patient lost their graft at 1 month due to early AMR and another The two cases of AMR in the eculizumab group occurred with a functioning graft died due to a bowel perforation at on posttransplant day 7 and 14, respectively, in the setting 7.8 months. Sixteen patients in the eculizumab group had of an increasing serum creatinine, increased DSA (BFXM 1-year follow-up and 50 in the control group had 1-year >400 and MFI>14 000), and a biopsy that showed both follow-up. No patient in the eculizumab group lost their C4d staining and glomerular microthrombi (13,14). PE graft in the first year. Graft survival at 1 year was 100% + was instituted resulting in the resolution of the histologic in the eculizumab group and 96% in the control group. features of AMR within 1 week, decreased serum DSA One-year protocol biopsy was obtained in 15 eculizumab- and a decrease in serum creatinine without the need for treated patients (1 refused) and 42 control patients (4 re- dialysis. fused and 3 were on anticoagulation, one had died and another had lost their graft early).

Antibody-mediated rejection Histology in the setting of high serum DSA levels The incidence of AMR in the first 3 months was 7.7% (2/26) Figure 2A shows typical changes of AMR in a control in the eculizumab group and 41.2% (21/51) in the histor- group patient with high levels of DSA. In addition to the ical control group (Table 2, p 0.0031) with all episodes light microscopic changes of AMR, the biopsy showed = occurring within 1 month of transplant. The percentage of C4d immunostaining of peritubular capillaries and ultra- + patients who developed high levels of DSA (B FXM >350 structural endothelial cell changes typical of AMR (loss of and/or MFI >10 000) in the first 3 months after transplan- fenestrations, cell enlargement, or microvillous changes in tation was similar in both groups (43% in controls and glomerular and peritubular capillaries). Figure 2B shows 50% in the eculizumab group, p 0.63). Evidence of typical microscopic findings in an eculizumab-treated pa- = proximal complement activation on biopsy (C4d immuno- tient with similar levels of DSA 5 days posttransplant. While + 2408 American Journal of Transplantation 2011; 11: 2405–2413 Stegall et al. HistologicalTerminal Complement Injury Inhibition Decreases Is Antibody-Mediated Ameliorated Rejection By Eculizumab

Figure 2: Continued.

Figure 2: Typical histologic appearance of renal allografts in control and eculizumab-treated patients who developedthe high biopsylevels was C4d , the light and electron microscopy ting of a rising serum creatinine (usually >2.0) and a rising of DSA after transplantation. Biopsies obtained in patients who had high levels of DSA (B flow crossmatch channel shift >350) at + the time of biopsy commonly showed histologic injury in control patients while no injury was present in eculizumab-treatedshowed patients normal features. serum DSA levels despite daily PE treatments. Splenec- despite evidence of early complement activation (C4d staining of peritubular capillaries). Panel A shows the typical appearance of tomy was performed in nine (17.7%) patients in the control + antibody-mediated rejection in a control group patient with a B-cell flow crossmatch level of 458 at the time of biopsy on postoperative group, including 43% (9/21) of the patients that developed day 11. Light microscopy (upper panel) reveals acute tubular injury, with dilatation of the tubules and flattening of the tubularOther epithelium early outcomes (black arrow), along with peritubular capillary inflammatory cell margination (white arrows) (hematoxylin and eosin). ImmunofluorescenceGraft dysfunction and the need for PE and AMR, but was not required in any eculizumab-treated pa- reveals diffuse, bright peritubular capillary staining for C4d (inset). By electron microscopy, both glomerular (left lower panel) and peritubular tients (p 0.025) including the patients that developed splenectomy: The eculizumab group generally demon- = capillary (right lower panel) endothelial cells show reactive changes, with enlargement of the cells and loss of fenestrations (arrowheads). AMR. Panel B shows the absence of histologic and ultrastructural changes in an eculizumab-treated patient with a B-cell flow crossmatchstrated level stable renal function during the first month. For of 553 at the time of biopsy on postoperative day 5. Light microscopy (upper panel) reveals normal-appearing glomeruli andexample, peritubular the mean increase in serum creatinine from the capillaries, without marginated inflammatory cells. No features of tubular injury are present. There is diffuse, bright peritubularposttransplant capillary nadir in the first month in the eculizumab Duration of eculizumab treatment and DSA levels staining for C4d (inset). By electron microscopy, endothelial cells in both glomerular and peritubular capillaries appear normal and show group was less than that of the control group (0.45 0.37 over time preserved fenestrations. ± mg/dL vs. 0.93 1.15, p 0.05, Table 2). By protocol, eculizumab was discontinued if the DSA ± = level was low (i.e. B flow crossmatch channel shift <200) Posttransplantation, 76% of patients in the control group at either 4 or 8 weeks. In the 16 patients followed for underwent posttransplant PE compared to only 12% in the 1 year, eight discontinued eculizumab at 4 weeks, six dis- American Journal of Transplantation 2011; 11: 2405–2413 2409 eculizumab group (Table 2, p < 0.0001). The first patient continued drug at 8 weeks, including one patient who de- transplanted in the eculizumab group developed high lev- veloped AMR. Two patients continued eculizumab for 1 els of DSA posttransplant despite daily PE therapy, yet year. No patient developed acute AMR after discontinua- the serum creatinine remained stable and biopsies did tion of eculizumab. not demonstrate AMR. Based on this case, the protocol was modified and the practice of routine, preemptive PE Chronic injury was discontinued. Indeed, 77% (10/13) of patients with a At 3, 6 and 12 months, the mean serum creatinine was high BFXM posttransplant did not undergo PE. The devel- similar in the historical control group and the eculizumab opment of severe AMR required aggressive intervention. group. Three eculizumab patients had serum creatinine Splenectomy was performed for severe AMR in the set- levels >2.0 mg/dL at 1-year follow-up. One patient had

2410 American Journal of Transplantation 2011; 11: 2405–2413 Long-term Outcomes in Patients Treated with Eculizumab Cornell et al: Am J Transplant 2015:15

From 2011-2016: 36 patients received tocilizumab 8 mg/Kg monthly for 6 -25 mo Maximal dose of 800 mg As ‘rescue therapy’ for cAMR with or without TG Am J Transpl 2017; XX:1-9

CNIs Uniquely Inhibit the Function of CD4+ Memory Cells Low immunological risk population: 1ary living donor Tx recipients rATG induction; tacro, MMF, pred >70% Caucasian recipients Median HLA MM 4 CTOT-09 – Tacro withdrawal

IdeS: IgG-degrading enzyme of S. pyogenes Cysteine proteinase that cleaves extracellular IgG with unique specificity in the hinge region

0.24 mg/Kg IBW0.24 mg/Kg0.24 mg/Kg IBW IBW